Printed, Flexible Lactate Sensors: Design Considerations Before Performing On-Body Measurements.

This work reports the process of sensor development, optimization, and characterization before the transition to on-body measurements can be made. Sensors using lactate oxidase as a sensing mechanism and tetrathiafulvalene as a mediator were optimized for sporting applications. Optimized sensors show linear range up to 24 mM lactate and sensitivity of 4.8 μA/mM which normalizes to 68 μA*cm-2/mM when accounting for surface area of the sensor. The optimized sensors were characterized 3 different ways: using commercially available reference and counter electrodes, using printed reference and counter electrodes, and using a printed reference electrode with no counter electrode. Sensors intended for measuring sweat must be selective in the presence of sweat constituents. Thus, in addition to traditional characterization in pH 7.0 buffer, we characterized sensor performance in solutions intended to approximate sweat. Sensor performance in pH 7.0 buffer solution was not reflective of sensor performance in artificial sweat, indicating that further characterization is necessary between sensor measurement in pH 7.0 buffer and on-body measurements. Furthermore, we performed enzyme activity measurements and sensor measurements concurrently in five different salts individually, finding that while NH4Cl and MgCl2 do not affect enzyme activity or sensor performance in physiologically relevant ranges of salt concentration, NaCl concentration or KCl concentration decreases enzyme activity and sensor current. On the other hand, CaCl2 induced a nonlinear change in sensor performance and enzyme activity with increasing salt concentration

Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo

The Endo16 gene of Strongylocentrotus purpuratus is expressed at the blastula stage of embryogenesis throughout the vegetal plate, at the gastrula stage in the whole of the archenteron and in postgastrular stages only in the midgut. We showed earlier that a 2300 bp upstream sequence suffices to faithfully recreate this pattern of expression when fused to a CAT reporter gene. Here we define the functional organization of this cis-regulatory domain, which includes over thirty high specificity binding sites, serviced by at least thirteen different putative transcription factors, in addition to >20 sites for a factor commonly found in the regulatory sequences of other sea urchin genes as well (SpGCF1). The Endo16 cis-regulatory domain consists of several different functional elements, or modules, each containing one or two unique DNA-binding factor target sites, plus sites for factors binding in other modules as well. Modular regulatory function was defined in experiments in which regions of the cis-regulatory DNA containing specific clusters of sites were tested in isolation, combined with one another, or by selective deletion, and the effects on expression of the CAT reporter were determined by whole-mount in situ hybridization or CAT enzyme activity measurements. The most proximal module (A) is mainly responsible for early embryonic expression, and module A alone suffices to locate expression in the vegetal plate and archenteron. The adjacent module (B) is responsible for a steep postgastrular rise in expression, when the gene is transcribed only in the midgut and, prior to this module B alone also suffices to promote expression in the vegetal plate and archenteron. The most distal module, G, acts as a booster for either A or B modules. However, no combination of A, B and G modules generates vegetal plate or gut expression exclusively. Ectopic expression of A-, B- and G-CAT fusion constructs occurs in the adjacent (veg1-derived) ectoderm and in skeletogenic mesenchyme cells. For expression to be confined to endoderm requires negative regulatory functions mediated by modules E, F and DC. Modules E and F each repress ectopic expression specifically in veg1 ectoderm. Module DC represses ectopic expression specifically in skeletogenic mesenchyme. Expression of some Endo16 constructs is dramatically increased by treatment with LiCl, which expands the territory in which the endogenous Endo16 gene is expressed at the expense of veg1 ectoderm. The same modules that act to repress ectopic expression in untreated embryos are required for enhanced expression of constructs after LiC1 treatment. Furthermore, both the negative spatial control functions and response to LiC1 require the presence of module A. The total regulatory requirements of the Endo16 gene during embryogenesis can be expressed in terms of the positive and negative functions of the individual modules and the interactions between modules that are identified in this study

Pathophysiological mechanisms in Parkinson`s Disease and Dystonia – converging aetiologies

In this thesis I used a range of experimental approaches including genetics, enzyme activity measurements, histology and imaging to explore converging pathophysiological mechanisms of Parkinson`s disease and dystonia, two conditions with frequent clinical overlap. First, based on a combined retro- and prospective cohort of patients, using a combination of lysosomal enzyme activity measurements in peripheral blood and brain samples, as well as a target gene approach, I provide first evidence of reduced levels of enzyme activity in Glucocerebrosidase and the presence of GBA mutations, indicating lysosomal abnormality, in a relevant proportion of patients with dystonia of previously unknown origin. Second, based on a retrospective cohort of patients, I detail that a relevant proportion of genetically confirmed mitochondrial disease patients present with a movement disorder phenotype - predominantly dystonia and parkinsonism. Analysing volumetric MRI data, I describe a patterned cerebellar atrophy in these particular patients. This also includes the first cases of isolated dystonia due to mitochondrial disease, adding the latter as a potential aetiology for dystonia of unknown origin. Third, I used a combination of post-GWAS population genetic approaches and tissue-based experiments to explore in how far the strong association between advancing age and Parkinson ́s disease is mediated via telomere length. Although the initial finding of an association between genetically determined telomere length and PD risk did not replicate in independent cohorts, I provide evidence that telomere length in human putamen physiologically shortens with advancing age and 3 is regulated differently than in other brain regions. This is unique in the human brain, implying a particular age-related vulnerability of the striatum, part of the nigro-striatal network, crucially involved in PD pathophysiology. I conclude by discussing the above findings in light of the current literature, expand on their relevance and possible direction of future experiments

Actinidin treatment and sous vide cooking : effects on tenderness and in vitro protein digestibility of beef brisket : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand

Actinidin from kiwifruit can tenderise meat and help to add value to low-value meat cuts. Compared with other traditional tenderisers (e.g. papain and bromelain) it is a promising way, due to its less intensive tenderisation effects on meat. But, as with other plant proteases, over-tenderisation of meat may occur if the reaction is not controlled. Therefore, the objectives of this study were (1) finding a suitable process to control the enzyme activity after desired meat tenderisation has been achieved; (2) optimising the dual processing conditions- actinidin pre-treatment followed by sous vide cooking to achieve the desired tenderisation in shorter processing times. The first part of the study focused on the thermal inactivation of actinidin in freshly-prepared kiwifruit extract (KE) or a commercially available green kiwifruit enzyme extract (CEE). The second part evaluated the effects of actinidin pre-treatment on texture and in vitro protein digestibility of sous vide cooked beef brisket steaks. The results showed that actinidin in KE and CEE was inactivated at moderate temperatures (60 and 65 °C) in less than 5 min. However, the enzyme inactivation times increased considerably (up to 24 h at these temperatures) for KE/CEE-meat mixtures, compared with KE/CEE alone. The thermal inactivation kinetics were used as a guide for optimising actinidin application parameters during the second phase of the study. For the final experiments, beef steaks were injected with 5 % (w/w, extract/meat) of CEE solution (3 mg/mL) followed by vacuum tumbling (at 4 °C for 15 min) and cooking (at 70 °C for 30 min) under sous vide conditions. This cooking time was considerably less than usual sous vide cooking times used in the meat industry. The actinidin-treated meat had no change in pH and colour, but showed a lower instrumental shear force; and improved sensory scores for tenderness, juiciness and flavour than the untreated meat steaks when tested by a sensory panel. Improved tenderness agreed well with the Transmission Electron Microscopy (TEM) results that showed considerable breakdown of the myofibrillar structure, particularly around the Z line. The addition of actinidin enhanced the rate of breakdown of muscle proteins, as shown by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and led to an increase in both protein solubility and ninhydrin-reactive free amino N release, during simulated gastric digestion. These results demonstrate the positive effects of actinidin on meat tenderness and meat protein digestibility during gastric digestion in vitro

Regulation of ethylene biosynthesis in Festuca novae-zelandiae (Hack.) Cockayne and in Festuca aruninaceae (Schreb.) in response to a water deficit : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

Changes in ethylene evolution and the associated biosynthetic enzyme ACC oxidase to a water deficit, were examined in intact leaves of Fostuca novae-zelandiae and F. arundinacea cultivar 'Roa' (syn. Schedonorus phoenix). The aim was to establish a role, or otherwise, for ACC oxidase as a regulator of ethylene biosynthesis in response to a water deficit. While ACC synthase has long been recognised as the major rate-limiting enzyme in ethylene biosynthesis, there is mounting evidence to suggest that ACC oxidase may also regulate the ethylene biosynthesic pathway in higher plants. Leaf tissues from the two species were harvested at regular intervals during the experimental dry-down, and dissected into two leaf zones, regions enclosed by the ligule. comprising the meristematic and elongating leaf zone (the enclosed tissue), and exposed regions composing the mature green leaf zones. Leaf proline content and the rate of leaf elongation (LER) were used as internal and external indicators of physiological changes in response to the water-deficit. Ethylene evolution in response to a water-deficit was found to be tissue-specific in F.arundinacea. In the rapidly expanding leaf zones, i.e. enclosed tissue, ethylene was maintained at levels similar to control tissue. In the mature green regions of leaves, ethylene followed changes in the leaf elongation rate (LER) with observed peaks in ethylene evolution occurnng approximately 48 hours after a rapid decline in the LER. This burst of ethylene was found to precede any accumulation of proline. Increases in the proline content in both leaf zones, only became significant after the ethylene evolution had subsided to below base levels. This stage-specific ethylene evolution in leaves suggests preferential protection of the rapidly expanding leaf cells, an observation that has been documented by other authors. ACO specific enzyme activity was greatest at soil water contents of ca. 9% in the enclosed and 10% in the exposed leaf tissues of F.arundinacea. On further purification of the enzyme, two novel proteins were recognised by polyclonal antibodies in water-stressed leaves of F.arundinacea. A 32 kDa protein was identified in the enclosed leaf tissue and a 37 kDa protein was identified in the exposed leaf tissue, by SDS-PAGE. These proteins eluted from a Mono Q column at different points in the separation process, i.e at salt concentrations of 320-340 and 300-320 mM NaCI respectively, indicating that they may represent two distinct isoforms of the ACO enzyme. Both proteins are active at pH 7.5 with saturating substrate (ACC) and co-substrate (Na ascorbate) concentrations of 1 mM and 30 mM respectively, and co-factor concentrations of 0.02 mM Fe² + and 30 mM NaHCO₃. When compared with results from western analyses, maximum specific enzyme activity correlated well with the water-deficit induced protein from partially purified enclosed leaf tissue, but only loosely with the protein identified in the exposed leaf tissue. The presence of high molecular weight proteins in both the crude and the purrfied (Mono Q) leaf extracts of F.arundinacea together with the novel proteins, suggests that the ACO enzyme in this species may exist as a dimer In F.novae-zelandiae, the presence of high molecular weight molecules m the crude and partially purified (Sephadex G-25) extracts also suggests dimensation of the enzyme in this species. From this study however, it is not possible to establish a clear regulatory role for the ACO enzyme in ethylene biosynthesis in either F.arvndinacea or F.novae-zelandiae While two novel water-deficit-induced proteins were associated with increased ACO activity in purified leaf extracts of F. amndinacea, there was no obvious correlation between ethylene evolution and enzyme activity

Tissue specific analysis reveals a differential organization and regulation of both ethylene biosynthesis and E8 during climacteric ripening of tomato

Background: Solanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed. Results: All tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature. Conclusions: The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening

What can one learn from two-state single molecule trajectories?

A time trajectory of an observable that fluctuates between two values (say, on and off), stemming from some unknown multi-substate kinetic scheme, is the output of many single molecule experiments. Here we show that when all successive waiting times along the trajectory are uncorrelated the on and the off waiting time probability density functions (PDFs) contain all the information. By relating the lack of correlation in the trajectory to the topology of kinetic schemes, we can immediately specify those kinetic schemes that are equally consistent with experiment, which means that it is impossible to differentiate between them by any sophisticated analyses of the trajectory. Correlated trajectories, however, contain additional information about the underlying kinetic scheme, and we consider the strategy that one should use to extract it. An example is given on correlations in the activity of individual lipase molecules.Comment: Biophys. J., in press (2005

Involvement of ethylene signalling in a non-climacteric fruit: new elements regarding the regulation of ADH expression in grapevine

Although grape berries have been classified as non climacteric fruits, ongoing studies on grape ethylene signalling lead to challenge the role of ethylene in their ripening. One of the significant molecular changes in berries is the up regulation of ADH (alcohol dehydrogenase, EC. 1.1.1.1) enzyme activity at the inception of fruit ripening and of VvADH2 transcript levels. This paper shows that the ethylene signal transduction pathway could be involved in the control of VvADH2 expression in grapevine berries and in cell suspensions. The induction of VvADH2 transcription, either in berries at the inception of ripening or in cell suspensions, was found to be partly inhibited by 1 methylcyclopropene (1 MCP), an inhibitor of ethylene receptors. Treatment of cell suspensions with 2 chloroethylphosphonic acid (2-CEPA), an ethylene releasing compound, also resulted in a significant increase of ADH activity and VvADH2 transcription under anaerobiosis, showing that concomitant ethylene and anaerobic treatments in cell suspensions could result in changes of VvADH2 expression. All these results, associated with the presence in the VvADH2 promoter of regulatory elements for ethylene and anaerobic response, suggest that ethylene transduction pathway and anaerobic stress could be in part involved in the regulation of VvADH2 expression in ripening berries and cell suspensions. These data open new aspects of the expression control of a ripening-related gene in a non climacteric fruit